Actn3 genotype screen for athletic performance

ABSTRACT

The present invention concerns novel methods of selecting or matching a sport or sporting event to an individual (e.g. a sprint/power sport or an endurance sport) and predicting athletic performance, the methods involving assessing ACTN3 genotype. In alternative embodiments, training regimens may be optimally designed for athletes by assessing the ACTN3 genotypes. Certain embodiments concern combining the assessment of the ACTN3 genotype with other known fitness-related genes to better assess the athletic potential of an individual. In addition, the genotypic analysis of the ACTN3 gene may be combined with physiological tests, physical measurements and/or psychological assessments to more optimally design a training regimen for an individual athlete.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 10/527,831filed Jan. 9, 2006, which claims the benefit of PCT Application No.PCT/AU2003/001202 (WO 2004/024947) filed Sep. 15, 2003, which claims thebenefit of Australian Patent Application No. 2002951411 filed Sep. 14,2002. Said applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for selecting or matching asport or sporting event to an individual (e.g., a sprint/power sport oran endurance sport) to increase their chances of success, optimizing thetraining programs of individuals, and for predicting the athleticperformance of individuals. Certain embodiments of the invention relateto identifying specific gene(s) or alterations in the gene(s) thatcorrelate with potential athletic performance. More particularly, theinvention relates to methods of genotyping an individual with respect tothe gene encoding the skeletal muscle protein, alpha-actinin-3 (ACTN3).In a specific embodiment, the ACTN3 genotype is determined for a singlenucleotide polymorphism (SNP) site 1747 C>T.

2. Description of Related Art

In an increasingly competitive environment for athletic performance,talent search programs are on the rise to ensure that those with thepotential to become an elite athlete are identified earlier in life toenable a head start in their efforts to reach their peak performance.These talent search programs are presently based on actual performancedata and phenotypic predictors determined by the type of training to beundertaken, as well as the likely demands from the particular sport. Oneweakness of both current training programs and talent search criteria isthe inability to determine whether an individual has already reachedhis/her performance potential, and so is unlikely to respond optimallyto further training.

Another weakness of the current talent search programs, which isparticularly relevant in countries with a relatively small populationbase in a large geographic area, is the opportunity for selection. Anindividual brought up in a environment with widespread access tosporting and coaching facilities is more likely to achieve success, andtherefore more likely to come to the attention of coaches and talentscouts than a young individual with potential who resides in arelatively isolated location or who might otherwise have anunderprivileged background. Similarly, individuals with potential toexcel in lower profile sports such as rowing may be overlooked simplybecause these sports programs are less available in most schools. Again,this diminishes the chances of early identification and participation,leading to subsequent overlook by coaches and talent scouts. These aredilemmas facing sporting organizations such as the Australian Instituteof Sport (AIS), since potential elite athletes are preferably selectedand inducted into relevant training programs at a young age.

The possibility exists that linkages or associations of genotype orgenotypic markers to certain physiological traits may contribute to orreduce performance in an elite athlete. Such methods may permit thedevelopment of DNA screens to assist in the selection of individualswith elite athlete potential. Such screens may help in overcoming someof the selection limitations of current talent search programs. Inaddition, such screening methods may assist in recognizing to whom andwhen a possibly small, but critical difference in an individual'straining program should be made.

The alpha-actinins are a family of actin-binding proteins related todystrophin and the spectrins (Blanchard, A. et al., Journal of MuscleResearch & Cell Motility, 10, 280-289, 1989). In skeletal muscle, thefamily members alpha-actinin-2 and alpha-actinin-3 are major structuralcomponents of sarcomeric Z-lines, where they function to anchoractin-containing thin filaments in a constitutive manner (Beggs, A. H.et al., Journal of Biological Chemistry, 267, 9281-9288, 1992). However,recent studies suggest additional roles for the alpha-actinins inskeletal muscle.

It has been found that sarcomeric alpha-actinins bind to other thinfilament and Z-line proteins including nebulin, myotilin, CapZ andmyozenin (Nave, R. et al., FEBS Letters, 269, 163-166, 1990, Papa, I. etal., Journal of Muscle Research & Cell Motility, 20, 187-197, 1999, andSalmikangas, P. et al., Human Molecular Genetics, 8, 1329-1336, 1999),the intermediate filament proteins, synemin and vinculin (Bellin, R. M.et al., Journal of Biological Chemistry, 274, 29493-29499, 1999, andMcGregor, A. et al., Biochemical Journal, 301, 225-233, 1994), and thesarcolemmal membrane proteins, dystrophin and .beta.1 integrin (Hance,J. E. et al., Archives of Biochemistry & Biophysics, 365, 216-222, 1999,and Otey, C. A. et al., Journal of Biological Chemistry, 268,21193-21197, 1993). These binding studies suggest that thealpha-actinins play a role in thin filament organization and theinteraction between the sarcomere cytoskeleton and the muscle membrane.In addition, sarcomeric alpha-actinin binds phosphatidylinositol4,5-bisphophate (Fukami, K. et al., Journal of Biological Chemistry,269, 1518-1522, 1994), phosphatidylinositol 3 kinase (Shibasaki, F. etal., Biochemical Journal, 302, 551-557, 1994) and PDZ-LIM adaptorproteins (Pomies, P. et al., Journal of Cell Biology, 139, 157-168,1997, and Pomies, P. et al., Journal of Biological Chemistry, 274,29242-29250), suggesting a role in the regulation of myo fiberdifferentiation and/or contraction.

In humans, the alpha-actinin-2 gene, ACTN2, is expressed in all skeletalmuscle fibers, while expression of ACTN3, encoding alpha-actinin-3, islimited to a subset of type 2 (fast) fibers (North, K. N. et al., NatureGenetics, 21, 353-354, 1999). It has been recently demonstrated thatalpha-actinin-3 is absent in about 18% of individuals in a range ofhuman populations and that homozygosity for a premature stop codon(577X) accounts for all cases of true alpha-actinin-3 deficiency statesidentified to date. An additional polymorphism (523R) occurs in linkagedisequilibrium with 577X, but does not appear to exert a deleteriouseffect when expressed in the heterozygous state in coupling with 577R.Further, absence of alpha-actinin-3 is not associated with an obviousdisease phenotype, suggesting that ACTN3 is redundant in humans (North,K. N. et al., 1999 Nature Genetics 21: 353-354).

Functional redundancy occurs when two genes perform overlappingfunctions so that inactivation of one of the genes has little or noeffect on the phenotype (reviewed in Nowak, M. A. et al., Nature, 388,167-171, 1997). In human skeletal muscle, alpha-actinin-2 expressioncompletely overlaps alpha-actinin-3 ACTN2 and ACTN3 are also 80%identical and 90% similar (Beggs, A. H. et al., 1992, supra), andalpha-actinin-2 and alpha-actinin-3 are capable of forming heterodimersin vitro and in vivo, suggesting structural similarity and lack ofsignificant functional differences between the two skeletal musclealpha-actinin isoforms (Chan, Y. et al., Biochemical & BiophysicalResearch Communications, 248, 134-139, 1998). It is hypothesised thatalpha-actinin-2 is able to compensate for the absence of alpha-actinin-3in type 2 (fast) fibers in humans.

SUMMARY OF THE INVENTION

Despite the apparent functional redundancy of ACTN3 and ACTN2 in humans,genotype screens of a pool of elite Australian athletes and notedCaucasian sprint athletes (particularly short distance runners, swimmersand cyclists) showed a very low frequency of homozygosity for the ACTN3premature stop codon 577X mutation (i.e. an ACTN3 null mutation, 577XX)relative to the Australian Caucasian population at large. It istherefore considered that screening for ACTN3 genotype, would provideconsiderable assistance in the selection of young individuals withpotential for elite performance in sprint-type sports and events. Also,the genotype screens showed that the frequency of the 577XX genotype wasrelatively higher in Caucasian elite endurance athletes. Thus, ascreening procedure for ACTN3 577XX genotype, may also provideassistance in identifying young individuals with potential for eliteperformance in endurance sports and events.

The present invention solves a need in the art by providing in vitromethods for screening individuals for athletic potential. In a oneembodiment, the genotype of an individual may be determined for the geneACTN3. In another embodiment, mRNA or protein is isolated from type 2skeletal muscle and analyzed for the presence or absence of ACTN3. Inanother embodiment, individuals are identified by isolating, DNA fromblood or buccal swab samples and the DNA is amplified and analyzed forthe presence or absence of a premature stop codon (577X) in the ACTN3gene. Other embodiments provide methods for screening individuals forathletic potential by combining the screening of ACTN3 with othergenetic or physiological tests. In addition, the methods describedprovide for developing training program(s) better suited for anindividual athlete by genetic assessments, physiological tests, physicalmeasurements and/or psychological assessments.

In another embodiment, the invention provides for screening individualsfor elite athletic potential, the method for example is carried out byobtaining a suitable muscle cell sample from an individual and detectingin the sample, alpha-actinin-3 protein and/or messenger RNA encodingthat protein.

Particular embodiments of the invention relate to a method of predictingthe presence or absence of a particular phenotype. The method comprisesobtaining a nucleic acid sample from an individual and determining theidentity of one or more bases (nucleotides) at specific (e.g.,polymorphic) sites of nucleic acid molecules described herein, whereinthe presence of a particular base at that site is correlated with aspecified phenotype, thereby predicting the presence, absence, orlikelihood of the presence or absence, of the phenotype in theindividual.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 illustrates the ACTN3 genotype frequency in controls, elitesprint/power athletes and elite endurance athletes.

TABLE 1: represents the genotypes of the R577X SNP in ACTN3 in Caucasianelite athletes of specific disciplines.

TABLE 2 represents a summary of individuals tested for number andfrequency (%) of ACTN3 alleles in controls and elite sprint/power andendurance athletes.

TABLE 3 represents SNPs identified in the ACTN3 gene thus far andcompiled in a list from the NCBI SNP website.

TABLE 4 represents symbols, full names, and cytogenic location ofnuclear and mitochondrial genes of the 200-\2 Human Gene Map forPerformance and Health-Related Fitness Phenotypes.

TABLE 5 represents endurance phenotypes and case-control studies (DNApolymorphisms).

TABLE 6 represents genotype and allele frequencies of ACTN3 577/R/Xalleles in human populations.

DEFINITIONS

As used herein in the specification, “a” or “an” may mean one or more.As used herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. “Eliteathlete” or variants thereof, refers to athletes that perform at thevery highest levels in terms of endurance, speed and/or strength (e.g.such that they are capable of competing at State, National and/orInternational levels in their sport).

As used herein, the terms “SNPs” or “single nucleotide polymorphisms”refer to single base changes at a specific location in an organism's(e.g., a human) genome.

DETAILED DESCRIPTION

In the following section, several embodiments of, for example, methodsare described in order to exemplify various embodiments of theinvention. It will be obvious though, to one skilled in the art thatpracticing the various embodiments does not require the employment ofall or even some of the specific details outlined herein. In some cases,well known methods or components have not been included in thedescription.

Methods and compositions to screen individuals for athletic potentialare disclosed. In one embodiment of the invention, a method to screenindividuals for the presence or absence of ACTN3 protein and/or mRNA isdisclosed. In another embodiment of the invention, a method to screenindividuals for the presence or absence of ACTN3 genotype variations isdisclosed. In another embodiment of the invention, a method to screenindividuals for the presence or absence of particular ACTN3 genotypes,such as 577RR, 577XR or 577XX is disclosed. Identification of ACTN3protein may be accomplished by directly measuring the protein levels orby indirectly measuring protein levels (e.g. antibodies etc).

ACTN3 Polymorphisms and Other Genetic Variations

A common polymorphism in humans has been identified in the gene encodingthe skeletal muscle protein, alpha-actinin 3 (ACTN3) that is onlypresent in type 2 (fast) fibers. Three possible genotypes 577RR (wildtype—expresses alpha-actinin-3), 577RX (heterozygous-alpha-actinin-3present), and 577XX (homozygous null—no alpha-actinin-3 in skeletalmuscle), have been identified. The allelic frequency varies in differentethnic groups (i.e. about 18% of Caucasians are alpha-actinin-3deficient compared to about 1% of African Zulus) (see Table 6 WestAfricans and African Americans). As discussed in the Examples below, inCaucasian elite sprint/power athletes, the frequency of the 577RRgenotype is very low. Thus a screening procedure for ACTN3 577XXgenotype, may provide assistance in identifying for example youngCaucasian individuals with potential for elite performance in sprint orpower-type sports and events. In contrast, in Caucasian elite enduranceathletes, the frequency of the 577XX genotype is relatively higher. Thusa screening procedure for ACTN3 577XX genotype, may also provideassistance in identifying for example young Caucasian individuals withpotential for elite performance in endurance sports and events. Inaddition, Table 6 illustrates the genotype and allele frequencies ofACTN3 577R/X alleles in different human populations. In Table 6 andTable 2, the negroid Africans (i.e. Zulus) screened have an extremelylow number of 577 XX individuals. Thus, the screening of ACTN3 innegroid African populations (and, likely, the related West Africans andAfrican-Americans) to detect 577XX genotypes may prove useful inidentifying individuals with sprint/power potential. In one embodiment,a method for screening for an ACTN3 allele (e.g. 577R, 577X) alone or incombination with another screening methods may be used to select, or atleast assist in the selection of, young individuals with elitesprint/power potential (e.g. potential as track sprinters, shortdistance swimmers, and track cyclists).

Other genes may also have beneficial effects on sprint/power and/orendurance athletic performance. For example, angiotensin-convertingenzyme (ACE) is reported to have two alleles, I and D, which have aneffect on athletic performance. The I allele is associated with lowerACE activity in both serum and tissue (Reider et al., “Sequencevariation in the human angiotensin converting enzyme.” Nat Genet, 1999vol. 22 pp 59-62). It is reported that there is an increased frequencyof the I allele in elite endurance athletes (Gayagay et al. 1998 “Eliteendurance athletes and the ACE I allele; the role of genes in athleticperformance”. Hum Genet 103:48-50; Montgomery et al. 1998 Human gene forphysical performance. Nature 393:221-222; Myerson et al. 1999 Humanangiotensin I-converting enzyme gene and endurance performance. J ApplPhysiol 87:1313-1316; Nazarov et al. 2001 The angiotensin convertingenzyme I/D polymorphism in Russian athletes Eur J Hum Genet 9:797-801).Conversely, an increased frequency of the ACE D allele has beenassociated with elite sprint performance (Myerson et al. 1999 Humanangiotensin I-converting enzyme gene and endurance performance. J ApplPhysiol 87:1313-1316; Nazarov et al. 2001 The angiotensin convertingenzyme I/D polymorphism in Russian athletes Eur J Hum Genet 9:797-801;Woods et al. 2001 Elite swimmers and the D allele of the ACE I/Dpolymorphism. Hum Genet 108: 230-232).

It is possible that there is a tradeoff between sprint and enduranceattributes that imposes limitations on the evolution of physicalperformance in humans and other vertebrates (Garland et al. 1990“Heritability of locomotor performance and its correlates in a naturalpopulation” Experientia 46:530-533). This is supported by data fromworld-class decathletes, which demonstrate that performance in the 100-msprint, shot-put, long-jump, and 110-m hurdles (relying on explosivepower and fast fatigue-susceptible muscle fibers) is negativelycorrelated with performance in the 1,500-m race (requiring endurance andfatigue-resistant slow fiber activity). (Van Damme et al. 2002Performance constraints in decathletes. Nature 415:755-756). Thissuggests that an individual may be predisposed toward specialistperformance in only one of the two areas (sprint/power vs. endurance).In particular embodiments of the invention, screening tests for ACTN3may be combined with one or more genetic tests for other performanceassociated genes. Such tests may include any gene that is known in theart to be associated with sprint/power and/or endurance performance(e.g., Rankinen et al. 2002, “The human gene map for performance andhealth-related fitness phenotypes: the 2001 update” Med. Sci. SportsExerc. 34: 1219-33; Perusse et al. 2003, “The human gene map forperformance and health-related fitness phenotypes: the 2002 update” Med.Sci. Sports Exerc. 35: 1248-1264 incorporated herein by reference intheir entirety).

Two reports (Rankinen et al. 2002; Perusse et al. 2003) have summarizedthe results of studies of performance and health-related fitnessphenotypes. A human performance and health-related fitness gene map isshown as FIG. 1 in the 2002 article. The map includes all gene entriesand QTL (quantitative trait loci) that have shown associations orlinkages with exercise-related phenotypes. The chromosomes and theirregions are from the Gene Map of the Human Genome web site, the NationalCenter for Biotechnology Information (NCBI), National Institutes ofHealth, Bethesda, Md. The loci abbreviations and full names of the genesof potential use in conjunction with ACTN3 screening are summarized inTABLE 4. In one embodiment, analysis of one or more of the genesreferenced in TABLE 4 may be used in combination with the evaluation ofthe ACTN3 gene of an individual to predict the elite athletic potentialof that individual.

TABLE 5 summarizes a study (Perusse et al., 2003) of alleles andgenotype frequencies of the ADRA2A (Alpha-2A-adrenergic receptor) andACE (Angiotensin 1 converting enzyme) genes between endurance athletesand sedentary controls. TABLE 5 illustrates the differences betweenendurance athletes and sedentary individuals. In one embodiment of theinvention, the examination of the ACTN3 genotype of a potential eliteathlete may be combined with the assessment of either the ADRA2Agenotype and/or the ACE genotype in order to more accurately predict theathletic potential of an individual. In another embodiment, theassessment of the ACTN3 genotype of an athlete may be combined with theassessment of either the ADRA2A genotype and/or the ACE genotype and/orother physiological assessments (e.g. VO₂ max etc.) to customize atraining regimen for the athlete.

Evolutionary Divergence of ACTN3 and ACTN2

Genotyping of non-human primates indicates that the 577X null mutationhas likely arisen in humans. The mouse genome contains four orthologueswhich all map to evolutionarily conserved regions for the four humangenes. Murine ACTN2 and ACTN3 are differentially expressed, spatiallyand temporally, during embryonic development, and in contrast to humans,alpha-actinin-2 expression does not completely overlap alpha-actinin-3in postnatal skeletal muscle, suggesting independent function.Furthermore, sequence comparison of human, mouse and chickenalpha-actinin genes demonstrates that ACTN3 has been conserved over along period of evolutionary time, implying a constraint on evolutionaryrate imposed by continued function of the gene. These observationsprovide a real framework in which to test theoretical models of geneticredundancy as they apply to human populations as well as other animals(Mills et al Differential Expression of the Actin-binding Proteins,alpha-actinin-2 and -3, in Different Species: Implications for theEvolution of Functional Redundancy” 2001 Hum Mol Gene 13:1335-1346).

To determine the origin of the 577X allele (and the 523R allele, whichoccurs in strong linkage disequilibrium with 577X), 36 unrelated baboons(diverged from human lineage 25×10⁶ years ago) and 33 unrelatedchimpanzees (diverged from human lineage 5×10⁶ years ago) weregenotyped. All 69 non-human primates were homozygous for the “wild-type”alleles in exons 15 (523Q) and 16 (577R), suggesting that thepolymorphisms originated after the separation of the human andchimpanzee lineages, or that they have a very low frequency in non-humanprimates (Mills et al 2001).

As for mice, the similarity between mouse ACTN2 and ACTN3 is the same asbetween human ACTN2 and ACTN3, i.e. 88% similar and 79% identical. Themouse proteins are collinear and have the same functional domains as thehuman proteins—an N-terminal actinin-binding domain, four central repeatdomains and C-terminal EF-hands (Mills et al 2001).

There is only one skeletal muscle ACTN gene in the chicken, whereas themouse genome contains four orthologues which all map to evolutionarilyconserved syntenic regions for the four human genes. Sequence comparisonbetween mouse and human ACTN2 and ACTN3 suggests that the evolution ofthe alpha-actinins has been slow relative to other genes. The low rateof substitution in ACTN3 appears not to be due to an intrinsically lowmutation rate in this gene (Mills et al 2001).

In other mammals, such as rabbits and pigs, there are also fast- andslow-muscle-specific iso forms of alpha-actinin, although the gene(s)responsible have not been isolated. The presence of two sarcomericalpha-actinin genes may, however, be restricted to mammals.

In mammals both copies of the gene have survived, and the comparison ofthe human and mouse ACTN2 and ACTN3 sequences shows that the genes havebeen highly conserved throughout mammalian evolution (Mills et al 2001).

Elite Athletic Performance and Horses

The horse is one of very few animals besides some dogs and camels thatis bred, kept or sold for its athletic performance and therefore isanother model for studying gene expression as it correlates withperformance. For example, the conservation of the ACTN3, an athleticmarker in humans for athletic potential, and ACTN2 gene throughoutspecies has been previously demonstrated. Although the equivalent genehas not yet been identified in horses, it is highly probable that a genelike ACTN3 exists in horses but has eluded detection. In certainembodiments of the invention, horses may be screened for an ACTN3-likegene. In other embodiments race horses such as the horses trained tocompete in a derby may be screened for an ACTN3-like gene.Alternatively, horses required to sprint with enormous power such aspolo ponies and barrel racing horses may also be screened fordifferential expression of an ACTN3-like gene. It is likely that thesprinting horses express a gene that is slightly different than anendurance horse and therefore analysis of the ACTN3-like gene may be anindicator of elite athletic potential in horses. Similar to what is seenin human athletes, screening a gene for a minor change, for example thepresence or absence of a specific nucleotide sequence (e.g. SNP site,deletion or insertion) may be a valuable indicator of elite athleticpotential in an animal such as a horse. An ACTN3-like gene is a genethat has the same function as the ACTN3 in other species and/or it hassequence similarities to the ACTN3 gene.

Previous studies indicate the equine angiotensin-converting enzyme genemight be a candidate gene for athletic performance in horses. The humanvariant of the gene contains a polymorphic marker that is associatedwith increased athletic ability of elite endurance athletes and anincreased anabolic response to training (Ellis et al, Characterizationof the Equine Angiotensin-converting Enzyme” 7th World Congress onGenetics Applied to Livestock Production, Aug. 19-23, 2002, Montpellier,France Session 05. Horse breeding Abstract of N^(o) 05-07 GENE. N. A. I.Tammen, F. W. Nicholas and H. W. Raadsma. ReproGen, University ofSydney, Camden, Australia). To date, a correlation in horses of the ACEexpression and elite athletic performance has been unsuccessful. Otherstudies including a study of the myosin heavy-chain gene (MyHC) inequine gluteus medius muscle where differential expression of the genehas been identified in foals but direct correlation of athleticabilities and presence or absence of the gene have not yet beencorrelated with performance (Eizema et al Differential Expression ofEquine Myosin heavy-chain mRNA and Protein Isoforms in a Limb muscle” JHistochem Cytochem 2003 September; 51 (9):1207-1216).

It is contemplated that the analysis of an ACTN3-like gene and otherphysiological and genetic parameters may be measured in horses in orderto more accurately access the elite athletic ability of a horse at anearly age. It is contemplated that horses may be pre-screened beforeusing them for breeding purposes to identify a more satisfactory geneticmatch. In addition it is possible that a foal in utero may be screenedin order to assess the athletic potential of the foal before it is born.The information generated from such screenings would save the breedersand investors of horses (camels, dogs) a tremendous amount of time andmoney as well as identify the potential ability of an animal at a earlystage of development. As with humans, the information generated fromgenotypic screening of a horse as well as other parameters (bloodlinesetc.) may help to identify a potential elite athlete and/or design abetter training regiment for a specific animal (e.g., a polo pony).

Single Nucleotide Polymorphisms (SNPs)

Various embodiments of the invention provide for methods for determininga correlation between a polymorphism or genetic variation (e.g, a SNP)and a phenotype, comprising: a) providing: samples from one or moresubjects; possibly medical records from one or more subjects, fordetermining a phenotype of the subject(s) and detection assays thatdetect a polymorphism; b) exposing the samples to detection assays underconditions such that the presence or absence of at least onepolymorphism is revealed; and; c) determining a correlation between theat least one polymorphism and the phenotype of the subjects.

Nucleic acids in the region of interest (e.g., the region containing thegenetic variation of interest) may be assayed using any suitable method,including but not limited to manual sequencing using radioactive markernucleotides, or automated sequencing. The sequence may be examined andthe presence or absence of a given SNP or mutation determined. Theparticular SNP site(s) (e.g. 1747 C>T of ACTN3) of a gene may be used toevaluate the presence, absence or change in a particular gene in orderto assess the athletic potential of an individual or modify a trainingregimen for that individual. The known SNPs for ACTN3 are listed inTABLE 3. In various embodiments of the invention, screening for the 1747C>T SNP of the ACTN3 gene may be combined with screening for any otherknown polymorphism in the ACTN3 gene, including but not limited to anySNP listed in TABLE 3.

Other SNPs of potential use in the practice of the claimed methods aredisclosed for example, in the Table of published U.S. patent applicationSer. No. 801274, publication No. 20020032319, incorporated herein byreference in its entirety. Any one or more of these sites may be assayedin combination with 1747 C>T SNP of the ACTN3 gene to predict theathletic potential of an individual, select or match a sport or sportingevent to an individual's chances of success) and/or to optimize atraining regimen.

In alternative embodiments of the invention, screening for geneticvariations may utilize other detection assays, such as anallele-specific hybridization assay. In a hybridization assay, thepresence of absence of a given SNP or other genetic variation isdetermined based on the ability of the DNA from the sample to hybridizeto a complementary DNA molecule (e.g., a oligonucleotide probe). Avariety of hybridization assays using a variety of techniques forhybridization and detection are known in the art and any such knowntechnique may be used in the claimed methods. Exemplary assays aredisclosed below.

In some embodiments, detection assays may utilize a DNA chiphybridization assay. In such assays, a series of oligonucleotide probesare affixed to a solid support. In some embodiments, the oligonucleotideprobes are designed to be unique to a given SNP or mutation. The DNAsample of interest is contacted with the DNA “chip” and hybridization isdetected. DNA chips, including customized DNA chips specific forparticular SNP sequences, are available from commercial sources such asAffymetrix (Santa Clara, Calif.).

In other exemplary embodiments, polymorphisms may be detected using aSNP-IT primer extension assay (Orchid Biosciences, Princeton, N.J.;e.g., U.S. Pat. Nos. 5,952,174 and 5,919,626). In this assay, SNPs areidentified by using a specially synthesized DNA primer and a DNApolymerase to selectively extend the DNA chain by one base at thesuspected SNP location. DNA in the region of interest is amplified anddenatured. Polymerase reactions are then performed using microfluidicsystems. Detection is accomplished by adding a label to the nucleotidesuspected of being at the SNP or mutation location. Incorporation of thelabel into the DNA can be detected by any suitable method (e.g., if thenucleotide contains a biotin label, detection is via a fluorescentlylabelled antibody specific for biotin). Other commercial kits may beused to identify the presence or absence of one or more SNPs (e.g.,Applied Biosystems: SNaPSOT, Assay-on-Demand, Assay-By-Design,Pyrosequencing.

Nucleic Acids

Various embodiments of the invention involve the isolation and analysisof nucleic acid molecules, such as DNA, mRNA or cDNA. Nucleic acids ofinterest may encode a portion or all of a targeted protein (e.g. ACTN3,ACE etc.). A “nucleic acid” as used herein includes single-stranded anddouble-stranded molecules, as well as DNA, RNA, chemically modifiednucleic acids and nucleic acid analogs. It is contemplated that anucleic acid within the scope of the present invention may be of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 275, about 300,about 325, about 350, about 375, about 400, about 425, about 450, about475, about 500, about 525, about 550, about 575, about 600, about 625,about 650, about 675, about 700, about 725, about 750, about 775, about800, about 825, about 850, about 875, about 900, about 925, about 950,about 975, about 1000, about 1100, about 1200, about 1300, about 1400,about 1500, about 1750, about 2000, about 2250, about 2500 or greaternucleotide residues in length, up to and including full-lengthchromosomal DNA.

Methods for partially or fully purifying DNA and/or RNA from complexmixtures, such as cell homogenates or extracts, are well known in theart. (See, e.g., Guide to Molecular Cloning Techniques, eds. Berger andKimmel, Academic Press, New York, N.Y., 1987; Molecular Cloning: ALaboratory Manual, 2nd Ed., eds. Sambrook, Fritsch and Maniatis, ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1989). Generally, cells,tissues or other source material containing nucleic acids are firsthomogenized, for example by freezing in liquid nitrogen followed bygrinding in a mortar and pestle. Certain tissues may be homogenizedusing a Waring blender, Virtis homogenizer, Dounce homogenizer or otherhomogenizer. Crude homogenates may be extracted with detergents, such assodium dodecyl sulphate (SDS), Triton X-100, CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate),octylglucoside or other detergents known in the art. As is well known,nuclease inhibitors such as RNase or DNase inhibitors may be added toprevent degradation of target nucleic acids.

Extraction may also be performed with chaotrophic agents such asguanidinium isothiocyanate, or organic solvents such as phenol. In someembodiments, protease treatment, for example with proteinase K, may beused to degrade cell proteins. Particulate contaminants may be removedby centrifugation or ultracentrifugation. Dialysis against aqueousbuffer of low ionic strength may be of use to remove salts or othersoluble contaminants. Nucleic acids may be precipitated by addition ofethanol at −20 degree C., or by addition of sodium acetate (pH 6.5,about 0.3 M) and 0.8 volumes of 2-propanol. Precipitated nucleic acidsmay be collected by centrifugation or, for chromosomal DNA, by spoolingthe precipitated DNA on a glass pipet or other probe. The skilledartisan will realize that the procedures listed above are exemplary onlyand that many variations may be used, depending on the particular typeof nucleic acid to be analyzed.

In certain embodiments, nucleic acids to be analyzed may be naturallyoccurring DNA or RNA molecules. Virtually any naturally occurringnucleic acid may be analyzed by the disclosed methods including, withoutlimit, chromosomal, mitochondrial or chloroplast DNA or ribosomal,transfer, heterogeneous nuclear or messenger RNA. Nucleic acids may beobtained from either prokaryotic or eukaryotic sources by standardmethods known in the art. Alternatively, nucleic acids of interest maybe prepared artificially, for example by PCR™ or other knownamplification processes or by preparation of libraries such as BAC, YAC,cosmid, plasmid or phage libraries containing nucleic acid inserts.(See, e.g., Berger and Kimmel, 1987; Sambrook et al., 1989.) The sourceof the nucleic acid is unimportant for purposes of analysis and it iscontemplated within the scope of the invention that nucleic acids fromvirtually any source may be analyzed.

Nucleic Acid Amplification

In particular embodiments, nucleic acids to be analyzed for screeningmay first be amplified to increase the signal strength. Nucleic acidsequences to be used as a template for amplification may be isolatedfrom cells contained in a biological sample (e.g. DNA or mRNA fromskeletal muscle), according to standard methodologies. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to convert the RNA to a complementary cDNA. In oneembodiment, the RNA is whole cell RNA and is used directly as thetemplate for amplification. In one example, the determination of theACTN3 genotype is performed by amplifying (e.g. by PCR) the ACTN3polynucleotide sequences, or more preferably a fragment thereof whichincludes the 1747 C>T SNP (e.g. exon 16), and sequencing theamplification products or otherwise detecting the presence and/orabsence of the 1747 C>T SNP in the amplification products. In anotherexample, it is known that the 577X allele contains a DdeI restrictionsite which can be readily detected by DdeI digestion of theamplification products and size fractionation of the digestion products(e.g. by gel electrophoresis). The size of the products may be used togenotype the ACTN3 locus in the individual. Various forms ofamplification are well known in the art and any such known method may beused. Generally, amplification involves the use of one or more primersthat hybridize selectively or specifically to a target nucleic acidsequence to be amplified.

Primers

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences may beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred. Methods of primerdesign are well-known in the art, based on the design of complementarysequences obtained from standard Watson-Crick base-pairing (i.e.,binding of adenine to thymine or uracil and binding of guanine tocytosine). Computerized programs for selection and design ofamplification primers are available from commercial and/or publicsources well known to the skilled artisan. Particular primer sequencesof use in detecting genetic variants predictive of athletic performance,such as the 1747 C>T SNP in ACTN3, are provided in the followingExamples. The skilled artisan will realize that the specific sequencesprovided are exemplary only and that alternative primer and/or probesequences may be used in the practice of the claimed methods.

Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given sample. One of the best knownamplification methods is the polymerase chain reaction (referred to asPCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202and 4,800,159.

One embodiment of the invention may comprise obtaining a suitable samplefrom an individual and detecting a specific messenger RNA, such as anACTN3 mRNA. An exemplary sample for use in this method is a muscletissue sample (e.g. muscle tissue biopsy, such as a punch biopsy). Oncethe tissue sample is obtained the sample may be prepared for isolationof the nucleic acids by standard techniques (e.g. single cell isolation,digestion of outer membranes, Oligo dT isolation of mRNA etc.) Theisolation of the mRNA may also be performed using kits known to the art(Pierce, AP Biotech, etc). A reverse transcriptase PCR amplificationprocedure may be performed in order to quantify an amount of mRNAamplified. Methods of reverse transcribing RNA into cDNA are well knownand described in Sambrook et al., 1989. Alternative methods for reversetranscription utilize thermostable DNA polymerases. These methods aredescribed in WO 90/07641 filed Dec. 21, 1990.

Another method for amplification of nucleic acids is the ligase chainreaction (“LCR”), disclosed in European Application No. 320 308. In LCR,two complementary probe pairs are prepared, and in the presence of thetarget sequence, each pair will bind to opposite complementary strandsof the target such that they abut. In the presence of a ligase, the twoprobe pairs will link to form a single unit. By temperature cycling, asin PCR, bound ligated units dissociate from the target and then serve as“target sequences” for ligation of excess probe pairs. U.S. Pat. No.4,883,750 describes a method similar to LCR for binding probe pairs to atarget sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., Proc. Nat'l Acad. Sci.USA 89:392-396, 1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases may be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencesmay also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA which is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still other amplification methods described in GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025 may be used in accordancewith the present invention. In the former application, “modified”primers are used in a PCR like, template and enzyme dependent synthesis.The primers may be modified by labelling with a capture moiety (e.g.,biotin) and/or a detector moiety (e.g., enzyme). In the latterapplication, an excess of labelled probes are added to a sample. In thepresence of the target sequence, the probe binds and is cleavedcatalytically. After cleavage, the target sequence is released intact tobe bound by excess probe. Cleavage of the labelled probe signals thepresence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR. Kwoh et al., Proc. Nat'l Acad. Sci. USA86:1173 (1989); Gingeras et al., PCT Application WO 88/10315. In NASBA,the nucleic acids may be prepared for amplification by standardphenol/chloroform extraction, heat denaturation of a clinical sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer which has target specificsequences. Following polymerization, DNA/RNA hybrids are digested withRNase H while double stranded DNA molecules are heat denatured again. Ineither case the single stranded DNA is made fully double stranded byaddition of second target specific primer, followed by polymerization.The double-stranded DNA molecules are then multiply transcribed by apolymerase such as T7 or SP6. In an isothermal cyclic reaction, theRNA's are reverse transcribed into double stranded DNA, and transcribedonce against with a polymerase such as T7 or SP6. The resultingproducts, whether truncated or complete, indicate target specificsequences.

Davey et al., European Application No. 329 822 disclose a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be usedin accordance with the present invention. The ssRNA is a first templatefor a first primer oligonucleotide, which is elongated by reversetranscriptase (RNA-dependent DNA polymerase). The RNA is then removedfrom the resulting DNA:RNA duplex by the action of ribonuclease H(RNaseH, an RNase specific for RNA in duplex with either DNA or RNA). Theresultant ssDNA is a second template for a second primer, which alsoincludes the sequences of an RNA polymerase promoter (exemplified by T7RNA polymerase) 5′ to its homology to the template. This primer isextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), producing a double-stranded DNA (“dsDNA”)molecule with a sequence identical to that of the original RNA betweenthe primers and having additionally, at one end, a promoter sequence.This promoter sequence may be used by the appropriate RNA polymerase tomake many RNA copies of the DNA. These copies may then re-enter thecycle leading to very swift amplification. With proper choice ofenzymes, this amplification may be done isothermally without addition ofenzymes at each cycle. Because of the cyclical nature of this process,the starting sequence may be chosen to be in the form of either DNA orRNA.

Miller et al., PCT Application WO 89/06700 disclose a nucleic acidsequence amplification scheme based on the hybridization of apromoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme is not cyclic, i.e., new templates are not produced from theresultant RNA transcripts. Other amplification methods include “race”and “one-sided PCR.” Frohman, M. A., In: PCR PROTOCOLS: A GUIDE TOMETHODS AND APPLICATIONS, Academic Press, N.Y. (1990) and Ohara et al.,Proc. Nat'l Acad. Sci. USA, 86:5673-5677 (1989).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. (e.g.,Wu et al., Genomics 4:560 1989).

Separation Methods

Following amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. (E.g., Sambrook et al., 1989) Alternatively, chromatographictechniques may be employed to effect separation. There are many kinds ofchromatography which may be used in the present invention (Freifelder,1982).

Identification Methods

Various methods for detection of nucleic acid sequence variants areknown in the art and any such known method may be used. In oneembodiment, detection may be by Southern blotting and hybridization witha labelled probe. The techniques involved in Southern blotting are wellknown to those of skill in the art (e.g., Sambrook et al., 1989).Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and non-covalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices. One example of the foregoing isdisclosed in U.S. Pat. No. 5,279,721, which shows an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is suited for carrying out methods accordingto the present invention.

Methods and apparatus for detecting nucleic acid sequence variants arecommercially available from a variety of sources, such as Third Wave,Pyrosequencing, Applied Biosystems, Affymetrix, Sequenom, Nanogen andothers and any such commercial system may be used to detect sequencevariants in ACTN3 or other performance related genes.

Proteins and Peptides

In certain embodiments, the disclosed methods may involve detectingand/or quantifying the amount of a specific protein (e.g. ACTN3) insamples to be screened. For convenience, the terms “protein,”“polypeptide” and “peptide are used interchangeably herein. Although avariety of methods of protein quantification are known in the art andmay be used, antibody-based assays, such as ELISA, are particularlyuseful for protein quantification. The skilled artisan will realize thatthe following discussion is exemplary only and that any known techniquesfor protein identification/quantification may be used.

In certain embodiments a protein or peptide may be isolated or purified.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, gel exclusionchromatography, HPLC (high performance liquid chromatography) FPLC (APBiotech), polyacrylamide gel electrophoresis, affinity chromatography,immunoaffinity chromatography and isoelectric focusing. An example ofreceptor protein purification by affinity chromatography is disclosed inU.S. Pat. No. 5,206,347, the entire text of which is incorporated hereinby reference. One of the more efficient methods of purifying peptides isfast performance liquid chromatography (FPLC) or even HPLC.

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

In certain embodiments, the disclosed methods may involve purifying oneor more proteins or peptides. It may be of use when purifying a proteinor a DNA sample that magnetic beads be used (Dynal, Dyna beads) toisolate the molecule and subsequently identify or quantitate the amountof molecule in a sample the molecule. These techniques are known bythose skilled in the art.

Antibodies

In certain embodiments, it may be desirable to make antibodies againstparticular proteins or peptides of interest (e.g. ACTN3). Theappropriate protein, or portions thereof, may be conjugated, orchemically linked to one or more agents to enhance their immunogenicity,as is well known in the art. Preferred agents are the carriers arekeyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).

In one embodiment, the detection of a targeted protein may be by Westernblot or immunocytochemistry using one or more specific antibodies to allor a portion of a target protein (e.g. ACTN3) with a specific antibodyor fragment thereof (e.g. Fab fragment or a recombinant antibodyfragment such as a scFv). One example of an antibody that may be used isanti-ACTN3 antibodies (as disclosed in North, K. N. et al.,Neuromuscular Disorders, 6, 229-235, 1996). In another embodiment, thelevel of a targeted protein may be detected by obtaining a sample froman individual (e.g. a muscle biopsy) and exposing the sample to one ormore antibodies directed to the targeted protein.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab', Fab, F(ab')₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. Techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

ELISA

In certain preferred embodiments, the amount of a protein of interest,such as ACTN3, may be determined by various types of enzyme linkedimmunosorbent assays (ELISAs) or radioimmunoassays (RIA) known in theart. Immunohistochemical detection using tissue sections is alsoparticularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and Western blotting, dotblotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, antibodies binding to the target proteins (e.g.ACTN3) are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a microtiter plate. A test compositionsuspected of containing the protein or portion of the protein isintroduced to the well. After binding and washing to removenon-specifically bound immune complexes, the bound antigen (protein ofinterest) may be detected. Detection is generally achieved by theaddition of a second antibody specific for the target protein that islinked to a detectable label. This type of ELISA is a “sandwich ELISA”.Detection may also be achieved by the addition of a second antibody,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label.

In another exemplary ELISA, the samples suspected of containing theprotein (antigen) are immobilized onto the well surface and thencontacted with the antibodies of the invention. After binding andwashing to remove non-specifically bound immune complexes, the boundantigen is detected. Where the initial antibodies are linked to adetectable label, the immune complexes may be detected directly.Alternatively, the immune complexes may be detected using a secondantibody that has binding affinity for the first antibody, with thesecond antibody being linked to a detectable label.

Another ELISA in which the proteins or peptides are immobilized,involves the use of antibody competition in the detection. In thisELISA, labelled antibodies are added to the wells, allowed to bind tothe target protein, and detected by means of their label. The amount oftarget antigen in an unknown sample is then determined by mixing thesample with the labelled antibodies before or during incubation withcoated wells. The presence of target antigen in the sample acts toreduce the amount of antibody available for binding to the well and thusreduces the ultimate signal. This is appropriate for detectingantibodies in an unknown sample, where the unlabelled antibodies bind tothe antigen-coated wells and also reduces the amount of antigenavailable to bind the labelled antibodies.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein and solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is more customary to use a secondary or tertiary detectionmeans rather than a direct procedure. Thus, after binding of a proteinor antibody to the well, coating with a non-reactive material to reducebackground, and washing to remove unbound material, the immobilizingsurface is contacted with the control biological sample to be testedunder conditions effective to allow immune complex (antigen/antibody)formation. Detection of the immune complex then requires a labelledsecondary binding ligand or antibody, or a secondary binding ligand orantibody in conjunction with a labelled tertiary antibody or thirdbinding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions mean that the incubation is at a temperatureand for a period of time sufficient to allow effective binding.Incubation steps are typically from about 1 to 2 to 4 hours, attemperatures preferably on the order of 25 C to 27 C, or may beovernight at about 4 C Or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact andincubate the first or second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labelled antibody, and subsequent to washingto remove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation (e.g., using avisible spectra spectrophotometer).

Kits

In still further embodiments, the present invention concerns detectionkits for use with the nucleic acid or immunodetection methods describedabove. Depending upon the type of assay to be utilized, a kit maycomprise one or more primer pairs for amplification of a target nucleicacid sequence, one or more probes, such as labelled probes, to detect agenetic variant, and one or more control target sequences to confirmamplification and/or probe binding conditions. Controls may include, forexample, specific target sequences for each allele of the 1747 C>T SNPin ACTN3. Probes may also be specific for hybridization to the 1747 C>TSNP alleles. Various other reagents of use, such as buffer, nucleotides,and polymerase may also be included.

In kits for immunoassay of protein, immunodetection kits may comprise,in suitable container means, a target protein or peptide, or a firstantibody that binds to a target protein or peptide, and animmunodetection reagent. The kits may comprise a first antibody specificfor the target protein or peptide and a labelled second antibodyspecific for the first antibody. Alternatively, kits may comprise afirst and a second antibody specific or selective for a protein ofinterest, with the second antibody labelled. Alternatively, the firstand second antibody may be unlabelled and a third antibody, specific forthe second antibody, may be included. Other standard reagents, such asbuffer and various substrates or reactants used to develop a labelledantibody may also be included.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich a sample may be placed, and preferably, suitably aliquoted. Wherea second or third binding ligand or additional component is provided,the kit will also generally contain a second, third or other additionalcontainer into which this ligand or component may be placed. Such kitsmay include injection or blow-molded plastic containers into which thedesired vials are retained.

Performance Testing

In certain embodiments, the screening methods of use may include, inaddition to ACTN3 assays, one or more performance based tests. Suchperformance tests may be used in combination with, for example, ACTN3SNP testing or ACTN3 protein or mRNA assays. Various exemplaryperformance tests are discussed below. The skilled artisan will realizethat the examples are not limiting and any performance assay known inthe art may be used.

VO₂ Max Testing

VO₂ max testing provides athletes with a direct measure of theirphysiological potential. Maximum oxygen consumption rates underconditions of vigorous exercise are determined by methods well known inthe art. Data includes aerobic and anaerobic thresholds, heart rate andspeed, ventilatory parameters, maximum heart rate and heart rate zones.

Anaerobic Threshold Testing (Blood Lactate & Ventilatory)

Anaerobic Threshold refers to the point in exercise where lactic acidproduction is equal to removal. This intensity is equivalent to a 60-120min run or cycle depending on fitness, technique and experience. Thetest is conducted by simultaneously measuring ventilation as well asblood lactate levels. Although the ventilatory and blood lactate methodsproduce very similar results, they both accurately determine anaerobicthreshold. Information provided by this test include blood lactatethreshold and ventilatory threshold, heart rates at anaerobic thresholdand speed (run) or watts (cycle) at anaerobic threshold

Anaerobic Power and Capacity Testing (Wingate Test)

The Wingate test determines leg power and capacity and is designed forpower sport athletes. The test is a 30 second all out effort on a cycleergometer that determines peak power and ability to resist fatigue. Datacollected from a Wingate test includes: (30 s test) peak power (watts),absolute, relative and fatigue index (how fast power drops off over the30 s test) and work (joules) (energy expenditure).

Critical Power (CP)

The goal of CP tests is to determine what is the optimal workload thatan athlete can sustain for a given time period or distance. The mostcommon CP tests may include CP (60-180 s), time frame dependant onsport; and CP Time Trial.

Resting Metabolic Rate (RMR)

RMR is also referred to as Resting Energy Expenditure (REE). It is anon-invasive method of determining the minimal amount of calories (Kcal)an individual utilizes in a day. The higher the RMR, the more caloriesan individual burns. The results are directly measured by O2 and CO2inspiration and expiration. One test protocol consists of no food oralcohol for 12 hours, no stimulants for 24 hours such as coffee and noexercise for 24-36 hours. The test is most commonly recommended forearly in the morning. The individual is connected to a metabolicmeasuring machine for 30 min while lying on his back in a rested state.During the test, the individual breathes into the metabolic measuringmachine through a mouthpiece and fitted hose. At the completion of thetest, the following information is gathered: Metabolic Rate(RMR)-Kcal/dayRespiratory Rate (RR), Respiratory Exchange Ratio (RER),Ventilation and heart rate at rest % of Carbohydrates and Fat utilizedat rest

Speed/Power Testing

Speed/Power Testing consists most commonly of three tests: RunningSpeed: Infrared Timing Lights (5-50 meters); a Vertical Jump & LegPower: Vertec apparatus and Agility Tests: Standard and Sport Specific.These tests assist in the analysis of an individuals capabilities in,for example, power sports).

Strength/Flexibility Testing

Strength/Flexibility testing generally consists of RM (resting muscle)strength: squat, bench, dead-lift, leg press; Muscular Endurance:repeated repetitions at a specified weight; Olympic Lifts Clean & Jerk,Snatch, Power Cleans, Power Snatch; Flexibility: standard andsport-specific and abdominal and lower back strength.

Body Composition

A body composition test may consist of a Harpenden skinfold caliper test(pinching the skin in several sites on the body such as under the arm,hip etc.) and estimating the percent body fat as well as estimating leanmuscle mass and fat mass. Another method involves immersion in water ina tank with deflated lungs. Body fat is measured by a special measuringdevice that determines water displacement.

Applicability of Methods

While the disclosed methods are suitable for the prediction of athleticperformance in sprint/power-type sports and events in Caucasianindividuals, the methods may also be suitable for use in any otherethnic group which generally shows a high frequency (i.e. preferably atleast 5%, more preferably at least 10%, and most preferably at least15%) of the 577XX genotype. After analyzing multiple Caucasians andseveral other ethnic groups, the null genotype if absent from anindividual athlete such as the Zulus and certain Caucasian femalesappears to correlate with the potential to be a sprint/power eliteathlete versus an endurance athlete. For example, the null genotype iscommon within the Native American population (29%), Asian population(25%) and White Europeans (20%), PNG Highlanders (15%), African Americanpopulation (13%) and the Aboriginal Australian population (10%).

Talent search programs may utilize the methods of the present inventionby themselves or in combination with similar methods for genotypingindividuals in respect of other genes linked to athletic performance.Other methods that may be combined with the methods disclosed are basedupon performance data and phenotypic predictors (e.g. height and build)and the like. Thus, the results of the methods of the present inventionmay be used to select, or at least assist in the selection of, youngindividuals with elite athlete potential and/or to provide guidance onthe type of sport that a young individual may choose to specialize.

In another embodiment, training programs may be devised for a potentialor current elite athlete that have greater chance of success, based onthe knowledge of genetic factors that will predict a person's trainingcapability (e.g. levels of ACTN3 protein or mRNA and/or SNP detection).Individualized training programs may focus on specific talents(determined from genetic makeup) by identifying the type of trainingthat is most likely to be successful. This would help to narrow thesmall margin between success and failure at the elite level, avoidunnecessary fatigue from excessive training without the expected gains(e.g. the genetic potential is not there); reduce wasted resources andpremature “burn out”; and may enhance long-term goals and self esteem inan individual athlete. Resources are wasted every time an individualwith elite athlete potential is removed from a program because he/shecannot achieve success. At a personal level, the effort and sacrificesalready undertaken by such individuals can adversely affect their lifegoals and self esteem. In these situations, knowledge of the geneticmakeup alone or in combination with other predictors may help to clarifywhy success has not been achieved, and will assist in directing theindividual to more realistic life goals that may include a moreappropriate sport.

Therefore, in one embodiment, identifying an improved training programfor an athlete may involve the determination of a specific genotype of atargeted gene (e.g. ACTN3 genotype) of an athlete. Another example ofdeveloping a training program for a potential or current athlete mayinvolve combining one or more tests for a targeted molecule with otherperformance assessing tests as indicated previously and analyzing theresults of the two or more tests to develop a program.

EXAMPLES Example 1 Screening for the ACTN3 Null (577XX) Genotype inElite Athletes Materials and Methods

Human genomic DNA was isolated from blood from a pool of elite athletes(108 endurance athletes and 83 sprint athletes), 88 African Zuluindividuals and 152 control Australian Caucasian individuals, byphenol:chloroform extraction following cell lysis with Triton-X100 anddigestion with proteinase K. Exon 16 of ACTN3 was amplified from genomicDNA. The primers corresponding to adjacent intronic sequences for exon16 were: TABLE-US-00001

forward 5′CTGTTGCCTGTGGTAAGTGGG3′ (SEQ ID NO: 1) reverse5′TGGTCACAGTATGCAGGAGGG3′ (SEQ ID NO: 2)

The PCR reaction cycle for the primers was: 35 cycles at 94 degree C.for 30 s and then 72 degree C. for 1 min, with a final extension of 94degree C. for 10 min. The R577X alleles (codons CGA and TGArespectively) can be distinguished by the presence (577X) or absence(577R) of a Dde I (C.dwnarw.TNAG) restriction site in Exon 16. 577R(wild type) PCR products have 205 by and 86 by fragments; while 577X PCRproducts have 108 bp, 97 by and 86 by fragments. Digested PCR fragmentswere separated by 10% polyacrylamide gel electrophoresis and visualizedby staining with ethidium bromide.

Results and Discussion

Results of the genotyping assays are shown in Table 2. ACTN3 genotypingwas conducted in elite athletes (i.e. individuals who perform at thehighest levels in terms of endurance, speed and/or strength). Comparedto controls, elite sprint athletes had a low frequency of the ACTN3 nullmutation 577XX (6% versus 18% in a control Caucasian population;p<0.05), similar to the trend observed in the Zulu population. Since,the force-generating capacity of type 2 muscle fibers at high velocity,the speed and tempo of movements, and the capacity of the individual toadapt to exercise training, all appear to be strongly geneticallyinfluenced, it is considered that ACTN3 genotype is likely to be afactor influencing normal variation in muscle function in the generalpopulation. Based on these results, ACTN3 genotyping is shown to be ofconsiderable potential in the selection, or at least to assist in theselection, of young individuals with elite athletic potential.

Example 2 Methods

436 unrelated Caucasian controls were genotyped from three differentsources (150 blood donors, 71 healthy children participating in anunrelated study, and 215 healthy adults participating in atalent-identification program with the Australian Institute of Sport),through use of the genotyping methodology described by Mills et al.(2001). Sex was known for 292 female controls and 134 male controls. 429elite Caucasian athletes were genotyped from 14 different sports. Forthe purposes of the example, Athletes were defined as “elite” if theyhad represented Australia in their sport at the international level; 50of the athletes had competed in Olympic Games.

Given the localization of alpha-actinin-3 in fast skeletal-musclefibers, it was hypothesized that deficiency of alpha-actinin-3 wouldreduce performance in sprint/power events and would therefore be lessfrequent in elite sprint athletes. To test this hypothesis, thegenotypes of a subset of 107 elite athletes (72 male and 35 female) wereanalyzed, classified a priori as specialist sprint/power athletes,blinded to genotyping results. This group comprised 46 track athletescompeting in events of 800 m, 42 swimmers competing in events of 200 m,9 judo athletes, 7 short-distance track cyclists, and 3 speed skaters.For comparison, a subset of 194 subjects (122 male and 72 female)classified independently as specialist endurance athletes and analyzed,including 77 long-distance cyclists, 77 rowers, 18 swimmers competingover distances of 400 m, 15 track athletes competing in events of 5,000m, and 7 cross-country skiers. Thirty-two sprint athletes (25 male and 7female) and 18 endurance athletes (12 male and 6 female) had competed atthe Olympic level. Because of the stringency of the classificationcriteria, 128 of the elite athletes could not be unambiguously assignedinto either the sprint/power or endurance groups and were excluded fromsubsequent analyses.

To test for homogeneity of ACTN3 allele and genotype frequencies betweenathlete and control groups, the log-linear modeling approach was used asdescribed by Huttley and Wilson (2000), implemented in the statisticalprogramming language R (version 1.6.2), through use of a package(contributed by J. Maindonald; available from The R Project forStatistical Computing Web site). “X” 2 values were estimated usinggenotype numbers for comparisons between athletes and controls. Thegenotypic profiles of the three control groups (150 blood donors, 71healthy children, and 215 healthy adults) did not differ significantlyfrom one another (x²=0.19; P=0.996) nor from a previously genotypedgroup of 107 white Europeans (Mills et al. 2001), suggesting that thegenotype frequencies in the control cohort are representative of abroader Caucasian population. ACTN3 genotype frequencies did not varysignificantly between male and female control subjects, and, overall,there was no significant deviation from Hardy-Weinberg (H-W)equilibrium.

ACTN3 genotyping data from the control, sprint/power, and endurancegroups are summarized in TABLE 2 and FIG. 1. There were no significantallele or genotype frequency differences between the elite athlete groupas a whole and the controls. However, when the athletes were dividedinto sprint/power and endurance groups and compared with controls, therewas strong evidence of allele frequency variation (x² _([df=5])=23;P<0.001) There were significant allele frequency differences betweensprint athletes and controls for both males (x² _([df=1])=14.8; P<0.001)and females (x² _([df=1])=7.2; P<0.01). Sprint athletes had a lowerfrequency of the 577XX (alpha-actinin-3 null) genotype (6% vs. 18%), andno female elite sprint athletes or sprint Olympians were 577XX. Thesprint athlete group also had a higher frequency of the 577RR genotype(50% vs. 30%) and a lower frequency of the heterozygous 577RX genotype(45% vs. 52%), compared with controls. Elite endurance athletes had aslightly higher frequency of the 577XX genotype (24%) than did controls(18%). More importantly, allele frequencies in sprint and enduranceathletes deviated in opposite directions and differed significantly fromeach other in both males (x² _([df=1])=13.3; P<0.001) and females (x²_([df=1])5.8; P<0.05). The differences between the two groupseffectively cancelled each other out, explaining the lack of associationwhen the entire elite athletic cohort was compared with the controlgroup.

Overall, there was also evidence of genotype variation that is notexplained by allele frequency differences (x² _([df=5])=16.7; P<0.01).This suggested variation in H-W disequilibrium coefficients amonggroups, despite there being no evidence for departure from H-Wequilibrium overall. The effect was restricted to female sprint (x²_([df=1])=7.4; P<0.01) and endurance (x² _([df=1])=6.0; P<0.05)athletes, with more heterozygous female sprint athletes than expected atH-W equilibrium (20 vs. 15) and fewer than expected heterozygous femaleendurance athletes (25 vs. 36). The allele-frequency-independentgenotype differences between female sprint and endurance athletes werehighly significant (x² _([df=1])=13.8; P<0.001). No effect was seen inmales, suggesting that the effect of ACTN3 genotype on performancediffers between males and females.

These findings suggest that the ACTN3 577R allele provides an advantagefor power and sprint activities. No female elite sprint athletes in thesample were alpha-actinin-3 deficient (compared with 8% of males). Inmales, the androgen hormone response to training is likely to make asignificant contribution to improvements in performance, so that therelative effect of alpha-actinin-3 on muscle power may be reduced.Interestingly, all male Olympian power athletes in the cohort had atleast one copy of the functional 577R allele of ACTN3 (associated withthe presence of alpha-actinin-3 in skeletal muscle), suggesting that“every variable counts” at the highest levels of sporting competition.Although at least 73 genetic loci have been associated with fitness andperformance phenotypes (Rankinen et al. 2002 “The human gene map forperformance and health-related fitness phenotypes: the 2001 update”. MedSci Sports Exerc 34:1219-1233), ACTN3 is the first structuralskeletal-muscle gene for which such an association has beendemonstrated.

The alpha-actinin-3 protein may promote the formation of fast-twitchfibers or alter glucose metabolism in response to training. In addition,alpha-actinin-3 may be evolutionarily optimized for the minimization ofdamage caused by eccentric muscle contraction. The Z line in fast,glycolytic fibers is the structure most vulnerable to exercise-inducedinjury resulting in morphological damage and degradation of associatedproteins, including the alpha-actinins (Friden and Lieber 2001,“Eccentric exercise-induced injuries to contractile and cytoskeletalmuscle fiber components Acta Physiol Scand 171:321-326).

If the 577XX genotype enhances endurance performance as the 577R alleleappears to enhance sprint-ability, then the 577R and 577X alleles may bemaintained in the population because they both confer selectiveadvantages under different environmental conditions and are thus kept athigh population frequencies by balancing selection.

Example 3

FIG. 1 represents a histogram compilation of ACTN3 genotype frequency incontrols, elite sprint/power athletes, and endurance athletes. Comparedwith healthy Caucasian controls, there is a marked reduction in thefrequency of the ACTN3 577XX genotype (associated with alpha-actinin-3deficiency) in elite Caucasian sprint athletes; remarkably, none of thefemale sprint athletes or sprint athletes who had competed at theOlympic level (25 males and 7 females) were alpha-actinin-3 deficient.Conversely, there is a trend toward an increase in the 577XX genotype inendurance athletes, although this association reaches statisticalsignificance only in females. Error bars indicate 95% CIs.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it areapparent to those of skill in the art that variations may be applied tothe COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itare apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

TABLE 1 Number and Frequency (%) of ACTN3 Genotypes and Frequency (%) ofACTN3 Alleles in Controls and Elite Sprint/Power and Endurance AthletesALLELE No. (%) FREQUENCY WITH GENOTYPE (%) GROUP (n) RR RX XX R X Male:Controls (134) 40 (30) 73 (54) 21 (16) 57 43 Sprint (72) 38 (53) 28 (39)6 (8) 72 28 Endurance (122) 34 (28) 63 (52) 25 (20) 54 46 Female:Controls (292) 88 (30) 147 (50)  57 (20) 55 45 Sprint (35) 15 (43) 20(57) 0 (0) 71 29 Endurance (72) 26 (36) 25 (35) 21 (29) 53 47 Total:Controls (436) 130 (30)  226 (52)  80 (18) 56 44 Sprint (107) 53 (50) 48(45) 6 (6) 72 28 Endurance (194) 60 (31) 88 (45) 46 (24) 54 46

TABLE 2 Genotyping of R577X in ACTN3 in Caucasians Elite Athletes. SportTotal 577RR 577RX Strength Sport ID Institute Number (%) (%) 577XX (%)Endurance Rower RT492- AIS 64 22 28 14  RT556 (34.4%) (43.8%) (21.8%)Endurance Triathloner RT977- AIS 13  3  8 2 RT989 (23.1%) (61.5%)(15.4%) Endurance Cyclist RT990- AIS 9  4  2 3 RT998 (44.4%) (22.2%(33.3%) Endurance Track KN246- AIS 22  7  7 8 Cyclist KN275 (31.8%)(31.8%) (36.4%) Endurance Marathon KN310 AIS 1  0  0 1 Endurance Allabove AIS 108 36 45 27  (33.3%) (41.7%) (25.0%) Sprint Swimmer RT901-AIS 45 17 25 3 RT1018 (37.8%) (55.6%)  (6.6%) Sprint Track KN246- AIS 8 4  3 1 Cyclist KN275 (50.0%) (37.5%) (12.5%) Sprint Athletics KN276-AIS 30 16 13 1 KN309 (53.3%) (43.3%)  (3.3%) Sprint All above AIS 83 3741 5 (44.6%) (49.4%)  (6.0%) Africa Zulu 88 69 18 1 (78.4%) (20.5%) (1.1%)* Australian 152 46 78 28  Caucasian (30.0%) (52.0%)   (18%)Control

TABLE 3 SNPs identified in the ACTN3 gene to date NCBI SNP CLUSTER IDrs2229456 rs2229455 rs2229454 rs2228325 rs1126675 rs7949754 rs7924602rs5792393 rs4990284 rs4990283 rs4013815 rs3937320 rs3837428 rs3814736rs3814735 rs3782080 rs2511217 rs2511216 rs2509559 rs2509558 rs2305537rs2305534 rs2290463 rs2275998 rs2096583 rs2000939 rs1815739 rs1791690rs1671064 rs679228 rs678397 rs677488 rs647476 rs647029 rs618838 rs607736rs597626 rs544021 rs540874 rs538330 rs531490 rs509556 rs490998 rs13897rs4576 rs1189338 rs1201433 rs640213 rs3737525 rs3178740 rs3180065rs3180064 rs3180063 rs3867132 rs608504 rs610293 rs3825065

TABLE 4 Symbols, full names and cytogenic location of nuclear andmitochondrial genes of the 2002 Human Gene Map for Performance andHealth-Related Fitness Phenotypes. Gene or Locus Name Location A BACADVL Acyl coenzyme A dehydrogenase, very long chain 17p13-p11 ACEAngiotensin I converting enzyme 17q23 ADRA2A Alpha-2A-adrenergicreceptor 10q24-q26 ADRB1 Adrenergic, beta-1-, receptor 10q24-q26 ADRB2Beta-2-adrenergic receptor 5q31-q32 ADRB3 Beta-3-adrenergic receptor8p12-p11.2 AGT Angiotensinogen 1q42-q43 ANG Angiogenin, ribonuclease,RNase A family, 5 14q11.1-q11.2 APOE Apolipoprotein E 19q13.2 ATP1A2ATPase, Na_/K_transporting, alpha-2 polypeptide 1q21-q23 ATP1B1 ATPase,Na_/K_transporting, beta I polypeptide 1q22-q25 BDKRB2 Bradykininreceptor B2 14q32.1-q32.2 C D E F G CASO2 Calsequestrin 2 (cardiacmuscle) 1p13.3-p11 CFTR Cystic fibrosis transmembrane conductanceregulator, ATP-binding cassette(sub-family C, member 7) 7q31.2 CKMCreatine kinase, muscle 19q13.2-q13.3 CNTF Ciliary neurotrophic factor11q12.2 CPT2 Carnitine palmitoyltransferase 2 1p32 COL1A1 Collagen, typeI, alpha 1 17q21.3-q22.1 EDN1 Endothelin 1 6p24.1 ENO3 Enolase 3, (beta,muscle) 17pter-p11 FABP2 Fatty acid binding protein 2 4q28-q31 FGAFibrinogen, A alpha polypeptide 4q28 FGB Fibrinogen, B beta polypeptide4q28 GDF8 (MSTN) Growth differentiation factor 8 (myostatin) 2q32.2 GNB3Guanine nucleotide binding protein (G protein), beta polypeptide 3 12p13H I K L M HLA-A Major histocompatibility complex, class I, A 6p21.3 HPHaptoglobin 16q22.1 IGF1 Insulin-like growth factor 1 12q22-q23 IGF2Insulin-like growth factor 2 11p15.5 IL-6 Interleukin-6 KCNQ1K_voltage-gated channel, KQT-like subfamily, member 1 11p15.5 LDHALactate dehydrogenase A 11p15.4 LPL Lipoprotein lipase 8p22 MTCO1Cytochrome c oxidase I mtDNA 5904-7445 MTCO3 Cytochrome c oxidase IIImtDNA 9207-9990 MTCYB Cytochrome b mtDNA 14747-15887 MTND1 NADHdehydrogenase 1 mtDNA 3307-4262 MTND4 NADH dehydrogenase 4 mtDNA10760-12137 MTND5 NADH dehydrogenase 5 mtDNA 12337-14148 MTTE TransferRNA, mitochondrial, glutamic acid mtDNA 14674-14742 MTTI Transfer RNA,mitochondrial, isoleucine mtDNA 4263-4331 MTTK Transfer RNA,mitochondrial, lysine mtDNA 8295-8364 MTTL1 Transfer RNA, mitochondrial,leucine 1 (UUR) mtDNA 3230-3304 MTTL2 Transfer RNA, mitochondrial,leucine 2 (CUN) mtDNA 12266-12336 MTTM Transfer RNA, mitochondrial,methionine mtDNA 4402-4469 MTTT Transfer RNA, mitochondrial, threoninemtDNA 15888-15953 MTTY Transfer RNA, mitochondrial, tyrosine mtDNA5826-5891 MyHC myosin Heavy-chain N O P Q R S T U V NOS3 Nitric oxidesynthase 3 (endothelial cell) 7q36 NPY Neuropeptide Y 7p15.1 PAI1Plasminogen activator inhibitor 1 7q21.3-q22 PFKM Phosphofructokinase,muscle 12q13.3 PGAM2 Phosphoglycerate mutase 2 (muscle) 7p13-p12 PGK1Phosphoglycerate kinase 1 Xq13 PHKA1 Phosphorylase kinase, alpha 1(muscle) Xq12-q13 PON1 Paraoxonase 1 7q21.3 PPARA Peroxisomeproliferative activated receptor, alpha 22q13.31 PPARG Peroxisomeproliferative activated receptor, gamma 3p25 PYGM Phosphorylase,glycogen, muscle 11q12-q13.2 RYR2 Ryanodine receptor 2 (cardiac)1q42.1-q43 SGCA Sarcoglycan, alpha (50 kDa dystrophin-associatedglycoprotein) 17q21 S100A1 S100 calcium binding protein A1 1q21 SURSulfonylurea receptor 11p15.1 TGFB1 Transforming growth factor beta 119q13.2 UCP2 Uncoupling protein 2 11q13 UCP3 Uncoupling protein 3 11q13VDR Vitamin D (1,25-dihydroxyvitamin D3) receptor 12q12-q14The gene symbols, names and cytogenetic locations are from the LocusLink web site available from the National Center for BiotechnologyInformation (NCBI). For mitochondrial DNA, locations are from the humanmitochondrial genome data base.

TABLE 5 Endurance phenotypes and case control studies (DNApolymorphisms). Athletes Gene Location N Sports Freq. N Freq. Controls PADRA2A 10q24-q26 140 Endurance 6.7/6.7: 0.77 141 6.7/6.7: 0.62 0.0376.7/6.3: 0.21 6.7/6.3: 0.34 6.3/6.3: 0.02 6.3/6.3: 0.04 6.7: 0.88 6.7:0.8 0.011 6.3: 0.12 6.3: 0.2 ACE 17q23 64 Endurance II: 0.30 118 II:0.18 0.03 ID: 0.55 ID: 0.51 DD: 0.16 DD: 0.32 I: 0.57 I: 0.43 0.02 D:0.43 D: 0.57 79 Running I: 0.57 Ref. I: 0.49 0.039 D: 0.43 Pop. D: 0.5125 Mountaineering NA Ref. NA 0.02 Pop. 0.003 60 Elite II: 0.25 Ref. II:0.16 0.009 athelets (cycling, running, handbull) ID: 0.58 Pop. ID: 0.45DD: 0.17 DD: 0.39 I: 0.54 I: 0.38 D: 0.46 D: 0.62 56 Elite II: 0.151248  II: 0.24 0.004 swimmers (subsample of 103 swimmers) ID: 0.39 ID:0.49 DD: 0.46 DD: 0.27 I: 0.34 I: 0.48 D: 0.66 D: 0.52 Reference:Perusse et al. 2003 “The human gene map for performance andhealth-related fitness phenotypes: the 2002 update” Med Sci. SportsExerc. 35: 1248-1264.

TABLE 6 Genotype and allelic frequencies of ACTN3 577R/X alleles inhuman populations. No. of Relative allele No. of genotypes frequency ofEthnic group chromosomes RX XX 577X Asian 56 14 7  0.5 ± 0.07 Javanese96 28 12 0.54 ± 0.05 Native American 14 2 2 0.43 ± 0.14 Asia/Americas166 44 21 0.52 ± 0.04 Hispanic 64 16 5 0.41 ± 0.06 White 214 47 21 0.42± 0.03 Europe 278 63 26 0.41 ± 0.03 Aboriginal 174 33 9 0.29 ± 0.03Australian PNG Highlander 78 16 6 0.36 ± 0.05 Australasia 252 49 15 0.31± 0.03 African American 90 12 6 0.27 ± 0.05 African Bantu 156 14 1 0.10± 0.05 Africa 246 56 7 0.16 ± 0.05 Unknown 152 50 11 0.47 Total 1094 23280

1-32. (canceled)
 33. A method to predict athletic performance in anindividual comprising: a) screening the individual for the presence ofone or more genetic variations in the alpha-actinin-3 (ACTN3) gene; andb) predicting athletic performance based on the presence of the one ormore genetic variations.
 34. The method of claim 1, wherein theindividual is a human, horse, dog or camel.
 35. The method of claim 1,comprising screening the individual for a 1747 C>T single nucleotidepolymorphism (SNP) in the ACTN3 gene.
 36. The method of claim 1,comprising genotyping the individual at the ACTN3 locus.
 37. The methodof claim 1, wherein the presence of at least one copy of the 577R alleleof the ACTN3 gene is positively associated with sprinting or powerperformance.
 38. The method of claim 1, wherein i) genotyping theindividual as a 577RR genotype is positively associated with sprintingor power performance, ii) genotyping the individual as a 577XX genotypeis negatively associated with sprinting or power performance, iii)genotyping the individual as a 577XX genotype is positively associatedwith endurance performance, iv) genotyping the individual as a 577RXgenotype is positively associated with sprinting or power performance infemale individuals, and v) genotyping the individual as a 577RX genotypeis negatively associated with endurance performance in femaleindividuals.
 39. The method of claim 1, comprising measuring the amountof ACTN3 protein present in the individual's skeletal muscle.
 40. Themethod of claim 7, wherein the amount of ACTN3 protein is measured usingan antibody specific for the ACTN3 protein.
 41. The method of claim 1,comprising measuring the amount of ACTN3 messenger RNA (mRNA) expressedin the individual's skeletal muscle.
 42. The method of claim 1,comprising identifying the 1747 C>T SNP alleles in the individual'sgenomic DNA by DNA sequencing, allele-specific hybridization,allele-specific amplification or restriction fragment lengthpolymorphism analysis.
 43. The method of claim 1, further comprisingscreening the individual for the presence of one or more geneticvariations in at least one other gene.
 44. The method of claim 11,wherein the at least one other gene is ACE (angiotensin-convertingenzyme) I allele, the ACE D allele, and/or ADRA2A (Alpha-2A-adrenergicreceptor) allele.
 45. The method of claim 12, wherein i) the ACE Iallele is positively associated with endurance performance, and ii) theACE D allele is positively associated with sprinting or powerperformance.
 46. The method of claim 1, further comprising screening theindividual using a test selected from the group consisting of VO2maximum, anaerobic threshold test, Wingate test, critical power, restingmetabolic rate, body composition, speed testing, power testing, strengthtesting, flexibility testing, muscle biopsy, fast twitch fiber test andslow twitch fiber test.
 47. A method of optimizing a training programcomprising: a) screening the individual for the presence of one or moregenetic variations in the α-actinin-3 (ACTN3) gene; and b) selecting theindividual's training program to optimize strength performance, powerperformance or endurance performance.
 48. A method of selecting a sportor sporting event for an individual comprising: a) screening theindividual for the presence of one or more genetic variations in theα-actinin-3 (ACTN3) gene; and b) selecting a sprint/power type sport orevent or, otherwise, and endurance sport or event on the basis of theresult of the said screening.